Method and apparatus for selectively targeting specific cells within a cell population

ABSTRACT

This invention provides a method and apparatus for selectively identifying, and targeting with an energy beam, specific cells within a cell population, for the purpose of inducing a response in the targeted cells. Using the present invention, every detectable cell in a population can be identified and affected, without substantially affecting non-targeted cells within the mixture.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.10/359,483, filed on Feb. 4, 2003, now U.S. Pat. No. 7,505,618, which isa continuation of U.S. patent application Ser. No. 09/728,281, filed onNov. 30, 2000, now U.S. Pat. No. 6,514,722, which is acontinuation-in-part of U.S. patent application Ser. No. 09/451,659,filed on Nov. 30, 1999, now U.S. Pat. No. 6,534,308, which is acontinuation-in-part of U.S. patent application Ser. No. 09/049,677,filed on Mar. 27, 1998, now U.S. Pat. No. 6,143,535, which is acontinuation-in-part of U.S. patent application Ser. No. 08/824,968,filed on Mar. 27, 1997, now U.S. Pat. No. 5,874,266. Each of theabove-mentioned applications and patents is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods and apparatus for selectivelytargeting specific cells within a population of living cells. Inparticular, this invention relates to high-speed methods and apparatusfor selectively identifying, and individually targeting with an energybeam, specific cells within a cell population to induce a response inthe targeted cells.

2. Description of the Related Art

The use of cellular therapies is growing rapidly, and is thereforebecoming an important therapeutic modality in the practice of medicine.Unlike other therapies, cellular therapies achieve a long-lasting, andoften permanent benefit through the use of living cells. Hematopoieticstem cell (HSC) (e.g. bone marrow or mobilized peripheral blood)transplantation is one example of a practiced, insurance-reimbursedcellular therapy. Many other cellular therapies are being developed,including immunotherapy for cancer and infectious diseases, chondrocytetherapy for cartilage defects, neuronal cell therapy forneurodegenerative diseases, and stem cell therapy for numerousindications. Many of these therapies require the removal of unwanted,detrimental cells for full efficacy to be realized.

Gene therapy is another active area of developing medicine that caninfluence the success of cellular therapy. Given the rapid advances inthe understanding of the human genome, it is likely that many genes willbe available for insertion into cells prior to transplantation intopatients. However, obtaining efficient targeted delivery of genes intospecific cells of interest has remained a difficult obstacle in thedevelopment of these therapies.

In the treatment of cancer, it has been found that high-dosechemotherapy and/or radiation therapy can be used to selectively killrapidly dividing cancer cells in the body. Unfortunately, several othercell types in the body are also rapidly dividing, and in fact, thedose-limiting toxicity for most anti-cancer therapies is the killing ofHSCs and progenitor cells in the bone marrow. HSC transplantation wasdeveloped as a therapy to rescue the hematopoietic system followinganti-cancer treatments. Upon infusion, the HSCs and progenitor cellswithin the transplant selectively home to the bone marrow and engraft.This process is monitored clinically through daily blood cell counts.Once blood counts return to acceptable levels, usually within 20 to 30days, the patient is considered engrafted and is released from thehospital.

HSC transplants have been traditionally performed with bone marrow, butmobilized peripheral blood (obtained via leukapheresis after growthfactor or low-dose chemotherapy administration) has recently become thepreferred source because it eliminates the need to harvest approximatelyone liter of bone marrow from the patient. In addition, HSCs frommobilized peripheral blood result in more rapid engraftment (8 to 15days), leading to less critical patient care and earlier discharge fromthe hospital. HSC transplantation has become an established therapy fortreating many diseases, such that over 45,000 procedures were performedworldwide in 1997.

HSC transplantation may be performed using either donor cells(allogeneic), or patient cells that have been harvested andcryopreserved prior to administration of high-dose anti-cancer therapy(autologous). Autologous transplants are widely used for treating avariety of diseases including breast cancer, Hodgkin's and non-Hodgkin'slymphomas, neuroblastoma, and multiple myeloma. The number of autologoustransplants currently outnumbers allogeneic transplants by approximatelya 2:1 ratio. This ratio is increasing further, mainly due tograft-versus-host disease (GVHD) complications associated withallogeneic transplants. One of the most significant problems withautologous transplants is the reintroduction of tumor cells to thepatient along with the HSCs, because these tumor cells contribute torelapse of the original disease.

As a tumor grows, tumor cells eventually leave the original tumor siteand migrate through the bloodstream to other locations in the body. Thisprocess, called tumor metastasis, results in the formation and growth ofsatellite tumors that greatly increase the severity of the disease. Thepresence of these metastatic tumor cells in the blood and other tissues,often including bone marrow, can create a significant problem forautologous transplantation. In fact, there is a very high probabilitythat metastatic tumor cells will contaminate the harvested HSCs that areto be returned to the patient following anti-cancer therapy.

The presence of contaminating tumor cells in autologous bone marrow andmobilized peripheral blood harvests has been confirmed in numerousscientific studies. Recent landmark studies have unambiguously shownthat reinfused tumor cells do indeed contribute to disease relapse inhumans (Rill et al. 1994). This was proven by genetically marking theharvested cells prior to transplant, and then showing that the markerwas detected in resurgent tumor cells in those patients who relapsedwith disease. These data have been confirmed by other investigators(Deisseroth et al. 1994), indicating that contaminating tumor cells inHSC transplants represent a real threat to patients undergoingautologous transplantation.

Subsequent detailed studies have now shown that the actual number oftumor cells reinfused in the transplant was correlated with the risk ofrelapse for acute lymphoblastic leukemia (Vervoordeldonk et al. 1997),non-Hodgkin's lymphoma (Sharp et al. 1992; Sharp et al. 1996), mantlecell lymphoma (Andersen et al. 1997), and breast cancer (Brockstein etal. 1996; Fields et al. 1996; Schulze et al. 1997; Vannucchi et al.1998; Vredenburgh et al. 1997). One of these studies went even further,showing that the number of tumor cells infused was inversely correlatedwith the elapsed time to relapse (Vredenburgh et al. 1997). These datasuggest that reducing the number of tumor cells in the transplant willlead to better outcomes for the patient.

Due to the known risk of tumor cell contamination in autologoustransplantation, a number of methods have been proposed for removingcontaminating tumor cells from harvested HSC populations. The basicprinciple underlying all purging methods is to remove or kill tumorcells while preserving the HSCs that are needed for hematopoieticreconstitution in the patient.

One such method utilized fluorescence-activated cell sorting (FACS) tosort HSCs from tumor cells (Tricot et al. 1995). As is known, flowcytometry sorts cells one at a time and physically separates onepopulation of cells from a mixture of cells based upon cell surfacemarkers and physical characteristics. However, it has been shown thatusing FACS to separate large cell populations for clinical applicationsis not advantageous because the process is slow, the cell yields can bevery low, and purity greater than 98% is rarely achieved.

Another method utilizing a flow cytometer is described in U.S. Pat. No.4,395,397 to Shapiro. In the Shapiro method, labeled cells are placed ina flow cytometer, and a downstream laser beam is used to kill thelabeled cells in the flowing stream after they pass by the detector andare recognized as being labeled by the electronic system. This methodsuffers from a number of disadvantages. Firstly, once an unwanted cellhas passed through the detector/laser region there is no way to checkthat destruction has been completed successfully. If a tumor cell evadesdestruction it will inevitably be reintroduced into the patient.Secondly, the focal spot diameter of the laser beam is of necessitygreater than the liquid stream cross section. Accordingly, many of theHSCs in the region of an unwanted cell will also be destroyed by thelaser beam. Also, as described above, the purity obtained by flowcytometric techniques is not very good due to the random and dynamicnature of a heterogeneous cell mixture that is flowing in a fast-moving(1-20 m/sec) stream of liquid.

Another method that utilizes laser technology is described in U.S. Pat.No. 4,629,687 to Schindler, et al. In this method, anchorage-dependentcells are grown on a movable surface, and then a small laser beam spotis scanned across the moving surface to illuminate cells one at a timeand the information is recorded. The same laser is then switched to ahigher lethal power level, and the beam is swept over the surface in allareas except where a cell of interest was recorded during theillumination step. Unfortunately, this method is slow and only will workon cells that can adhere to a surface.

A still further method that utilizes laser technology is described inU.S. Pat. No. 5,035,693 to Kratzer. In this method, cells are placed ona moving belt and a small laser beam spot is scanned across the surface.When a particular cell radiates in response to the illuminating laserspot, the same laser is quickly switched to high power in order to killthe cell in a near simultaneous manner before the scanner has movedappreciably away from that cell. However, this system has many of thesame disadvantages as the Shapiro method. For example, because thescanner is continuously moving during the imaging and killing of cells,the system is highly-dynamic, and therefore less stable and lessaccurate than a static system. Also, because the cells are moving on abelt past the detector in one direction, the method is not reversible.Thus, if a single tumor cell escapes detection, it will be reintroducedinto the patient.

Others have used a small laser beam spot to dynamically scan over asurface to illuminate cells. For example, U.S. Pat. No. 4,284,897 toSawamura et al. describes the use of galvanometric mirrors to scan asmall laser beam spot in a standard microscope to illuminate fluorescentcells. U.S. Pat. No. 5,381,224 to Dixon et al. describes imaging ofmacroscopic specimens through the use of a laser beam spot that israster-scanned with galvanometric mirrors through an F-theta scanninglens. In U.S. Pat. Nos. 5,646,411, 5,672,880, and 5,719,391 to Kain,scanning of a small laser spot with galvanometers through an F-thetalens is described. All of these imaging methods dynamically illuminate asmall point that is moved over the surface to be imaged. In some cases,the surface being scanned is also moving during imaging.

Similar methods of scanning a small laser spot have been described forpurposes other than imaging of cells. For example, U.S. Pat. No.4,532,402 to Overbeck describes the use of galvanometers to move a smalllaser beam spot over a semiconductor surface for repair of an integratedcircuit. Similarly, U.S. Pat. No. 5,690,846 to Okada et al. describeslaser processing by moving a small laser spot with mirrors through anF-theta scanning lens. U.S. Pat. No. 5,296,963 to Murakami et al.describes the use of galvanometric mirrors to scan a small laser beamspot in a standard inverted microscope to puncture cells for insertionof genetic matter.

Yet another method of scanning a biological specimen is described inU.S. Pat. No. 5,932,872 to Price. This method uses a plurality ofdetectors to simultaneously capture images at a plurality of focusplanes from a constantly moving surface. The resultant images can beused to choose the best-focus image in real-time, and can be used togenerate a three-dimensional volumetric image of a specimen.

Most of the methods described above are based on administering a tumorcell-removal or tumor cell-killing strategy to the entire harvested cellpopulation as a whole. In flow cytometry, cells are sorted on a singlecell basis to physically separate the unwanted tumor cells from HSCs.While each of these methods has been shown to reduce tumor cell numbersin HSC transplants, none has demonstrated the ability to remove or killall detectable tumor cells. In fact, the majority of patient transplantsstill contain detectable tumor cells after these purging techniques areused. Approximately 30 to 30,000 tumor cells per transplant stillremain, even after multiple-step purging procedures (Gazitt et al. 1995;Gribben et al. 1991; Mapara et al. 1997; Paulus et al. 1997). Further,all of these methods result in some degree of HSC loss or damage, whichcan significantly impact the success of the HSC transplant by delayingpatient engraftment. In summary, existing purging technologies areinadequate, and there exists a great unmet clinical need for novelapproaches that can effectively purge all detectable tumor cells from anHSC transplant. The method and apparatus described herein fulfills thisneed.

High throughput screening for the action of candidate drug compounds onbiological specimens is another area of great importance. Typically, alarge number of candidate compounds is applied in parallel to small cellsamples placed in wells of a multi-well plate, and each well is examinedfor some change in a biological indicator. Due to the large number ofcompounds, speed of screening is an important factor. Such studies arecurrently limited by capturing a signal from the cell population as awhole, or by laborious manual viewing of individual cells withmicroscopes. The former precludes the possibility of observing an effecton a cell subpopulation or of observing an effect within only a portionof the cell, whereas the latter approach is too slow to apply tonumerous candidate compounds. An apparatus and method that could rapidlymeasure the effect of candidate compounds on individual cells is ingreat need.

SUMMARY OF THE INVENTION

This invention provides a high-speed method and apparatus forselectively identifying, and individually targeting with an energy beam,specific cells within a cell population for the purpose of inducing aresponse in the targeted cells. Using the apparatus of the presentinvention, every detectable target cell in a cell population can bespecifically identified and targeted, without substantially affectingcells that are not being targeted. The cells can be a mixed populationor of a homogenous origin.

Specific cells are identified with the disclosed invention using severalapproaches. One embodiment includes a non-destructive labeling method sothat all of the cells of a first population are substantiallydistinguishable from the remaining cells of the cell mixture, theremaining cells comprising the second population. In this embodiment, alabeled antibody can be used to specifically mark each cell of the firstpopulation, yet not mark cells of the second population. The labeledcells are then identified within the cell mixture. A narrow energy beamis thereafter focused on the first of the targeted cells to achieve adesired response. The next of the targeted cells is then irradiated, andso on until every targeted cell has been irradiated.

In another embodiment, an antibody that selectively binds to cells ofthe second population, but not cells of the first population, is used toidentify cells of the first population. Cells of the first populationare identified by the absence of the label, and are thereafterindividually targeted with the energy beam.

The nature of the response that is induced by the energy beam isdependent upon the nature of the energy beam. The response can be lethalor non-lethal. Thus, examples of responses that can be induced with anenergy beam include necrosis, apoptosis, optoporation (to allow entry ofa substance that is present in the surrounding medium, including geneticmaterial), cell lysis, cell motion (laser tweezers), cutting of cellcomponents (laser scissors), activation of a photosensitive substance,excitation of a fluorescent reagent, photobleaching, and molecularuncaging.

Another aspect of the invention is directed to an apparatus fordetermining a morphological or physiological characteristic ofindividual cells in a biological specimen. Thus, the apparatus woulddirectly or indirectly induce a non-lethal response in a population ofcells, and measure the response. Such non-lethal responses includeoptoporation, cell motion (laser tweezers), cutting of cell components(laser scissors), activation of a photosensitive substance andexcitation of a fluorescent reagent. Such non-lethal responses may alsoinclude the responses of cells targeted by the techniques ofphotobleaching, such as photobleaching recovery, and molecular uncaging,both internal and external with respect to the cell. An example of suchapparatus would comprise:

An illumination source for illuminating a frame of cells in saidbiological specimen;

An image capture system that captures an image of said frame of cells;

First commands for determining the location of a first individual cellin said biological specimen by reference to said image;

An energy source that emits an energy beam sufficient to induce aresponse in at least one or more individual cells;

Second commands for intersecting said first individual cell with anenergy beam sufficient to interrogate said first individual cell for thepresence of a morphological or physiological characteristic; and

A detector for measuring the response of said first individual cell tosaid interrogation.

Alternatively, such an apparatus would comprise:

An illumination source for illuminating a frame of cells in saidbiological specimen;

An image capture system that captures an image of said frame of cells;

First commands for determining the location of a first targeted cell insaid biological specimen by reference to said image;

Second commands for intersecting said first targeted cell with an energybeam sufficient to cause a change of a morphological or physiologicalcharacteristic in said first targeted cell; and

A detector for measuring the response for the change in said morphologicor physiological characteristic of said first targeted cell.

Another aspect of the invention is an apparatus an illumination sourcefor illuminating a frame of cells in said biological specimencomprising, an image capture system that captures an image of said frameof cells, first commands for determining the location of an individualcell in said biological specimen by reference to said image, an energysource that emits an energy beam sufficient to induce a response in atleast one of the individual cells, second commands for intersecting saidfirst individual cell with an energy beam sufficient to cause a changeof a morphological or physiological characteristic in said firstindividual cell, and a detector for measuring a morphological orphysiological characteristic of said first individual cell.

Another aspect of the invention is an apparatus for determining amorphological or physiological characteristic of individual cells in abiological specimen comprising:

an illumination source for illuminating a frame of cells in saidbiological specimen, wherein said specimen contains a quantity of cagedcompounds;

an image capture system that captures an image of said frame of cells;

first commands for intersecting one or more of said quantity of cagedcompounds with an energy beam sufficient to uncage said one or more ofsaid quantity of caged compounds; and

an energy source that emits an energy beam sufficient to uncage one ormore of said quantities of caged compounds;

second commands to monitor the progress of the change in saidmorphological or physiological characteristic in response to saiduncaged compounds of said one or more of said quantity of cagedcompounds.

Another such apparatus for determining a morphological or physiologicalcharacteristic of individual cells in a biological specimen comprises:

an illumination source for illuminating a frame of cells in saidbiological specimen;

an image capture system that captures an image of said frame of cells;and first commands for determining the morphological or physiologicalcharacteristic of said individual cell in said frame of cells.

The above apparatus can be used for high throughput screening ofresponses of cells to outside stimuli, in that it provides rapidlocation and measurement of responses of individual cells to suchstimuli. Thus, the illumination source and/or the energy beam couldtarget molecules in the vicinity of the cells, whether or not themolecules are in or on the cells, the energy beam could interact withand activate the molecules and the illumination source could study theresponse of the cells to the molecules so activated.

A large number of commercially important research and clinicalapplications can be envisioned for such an apparatus, examples of whichare presented below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of one embodiment of a cell treatmentapparatus and illustrates the outer design of the housing and display.

FIG. 2 is a perspective view of one embodiment of a cell treatmentapparatus with the outer housing removed and the inner componentsillustrated.

FIG. 3 is a block diagram of the optical subassembly design within oneembodiment of a cell treatment apparatus.

FIG. 4 is a perspective view of one embodiment of an optical subassemblywithin one embodiment of a cell treatment apparatus.

FIG. 5 is a side view of one embodiment of an optical subassembly thatillustrates the arrangement of the scanning lens and the movable stage.

FIG. 6 is a bottom perspective view of one embodiment of an opticalsubassembly.

FIG. 7 is a top perspective view of the movable stage of the celltreatment apparatus.

DETAILED DESCRIPTION

A method and apparatus is described for selectively identifying, andindividually targeting with an energy beam, specific cells within a cellpopulation for the purpose of inducing a response in the targeted cells.The population of cells can be a mixed population or homogenous inorigin. The responses of any of the embodiments of the methods andapparatuses of the invention can be lethal or non-lethal. Examples ofsuch responses are set forth above and throughout this disclosure. Thecells targeted can be labeled as is often the case when the specimen isa mixed population. On the other hand, when the specimen is homogenous,the targeted cells can be those individual cells that are beinginterrogated or intersected by the illumination source or the energybeam, in order to study the response of the cell. For instance, suchresponses include the morphological or physiological characteristics ofthe cell. Generally, the method first employs a label that acts as amarker to identify and locate individual cells of a first population ofcells within a cell mixture that is comprised of the first population ofcells and a second population of cells. The cells targeted by theapparatus and methods herein are those that are selectively labeled, inthe case of a mixed population of cells, or the ones undergoinginterrogation or intersection by the illumination source or energy beam.

The chosen label can be any that substantially identifies anddistinguishes the first population of cells from the second populationof cells. For example, monoclonal antibodies that are directly orindirectly tagged with a fluorochrome can be used as specific labels.Other examples of cell surface binding labels include non-antibodyproteins, lectins, carbohydrates, or short peptides with selective cellbinding capacity. Membrane intercalating dyes, such as PKH-2 and PKH-26,could also serve as a useful distinguishing label indicating mitotichistory of a cell. Many membrane-permeable reagents are also availableto distinguish living cells from one another based upon selectedcriteria. For example, phalloidin indicates membrane integrity,tetramethyl rhodamine methyl ester (TMRM) indicates mitochondrialtransmembrane potential, monochlorobimane indicates glutathionereductive stage, carboxymethyl fluorescein diacetate (CMFDA) indicatesthiol activity, carboxyfluorescein diacetate indicates intracellular pH,fura-2 indicates intracellular Ca²⁺ level, and5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo carbocyanineiodide (JC-1) indicates membrane potential. Cell viability can beassessed by the use of fluorescent SYTO 13 or YO PRO reagents.Similarly, a fluorescently-tagged genetic probe (DNA or RNA) could beused to label cells which carry a gene of interest, or express a gene ofinterest. Further, cell cycle status could be assessed through the useof Hoechst 33342 dye to label existing DNA combined withbromodeoxyuridine (BrdU) to label newly synthesized DNA.

It should be noted that if no specific label is available for cells ofthe first population, the method can be implemented in an inversefashion by utilizing a specific label for cells of the secondpopulation. For example, in hematopoietic cell populations, the CD34 orACC-133 cell markers can be used to label only the primitivehematopoietic cells, but not the other cells within the mixture. In thisembodiment, cells of the first population are identified by the absenceof the label, and are thereby targeted by the energy beam.

After cells of the first population are identified, an energy beam, suchas from a laser, collimated or focused non-laser light, RF energy,accelerated particle, focused ultrasonic energy, electron beam, or otherradiation beam, is used to deliver a targeted dose of energy thatinduces the pre-determined response in each of the cells of the firstpopulation, without substantially affecting cells of the secondpopulation.

One such pre-determined response is photobleaching. In photobleaching, alabel in the form of a dye, such as rhodamine 123, GFP, fluoresceinisothiocyanate (FITC), or phycoerythrin, is added to the specimen beforethe instant methods are commenced. After the population of cells hastime to interact with the dye, the energy beam is used to bleach aregion of individual cells in the population. Such photobleachingstudies can be used to study the motility, replenishment, dynamics andthe like of cellular components and processes.

Another response is internal molecular uncaging. In such a process, thespecimen is combined with a caged molecule prior to the commencement ofthe instant methods. Such caged molecules include thebeta-2,6-dinitrobenzyl ester of L-aspartic acid or the1-(2-nitrophenyl)ethyl ether of 8-hydroxypyrene-1,3,6-tris-sulfonicacid. Similarly, caging groups including alpha-carboxy-2-nitrobenzyl(CNB) and 5-carboxymethoxy-2-nitrobenzyl (CMNB) can be linked tobiologically active molecules as ethers, thioethers, esters, amines, orsimilar functional groups. The term “internal molecular uncaging” refersto the fact that the molecular uncaging takes place on the surface orwithin the cell. Such uncaging experiments study rapid molecularprocesses such as cell membrane permeability and cellular signaling.

Yet another response is external molecular uncaging. This usesapproximately the same process as internal molecular caging. However, inexternal molecular uncaging, the uncaged molecule is not attached to orincorporated into the targeted cells. Instead, the responses of thesurrounding targeted cells to the caged and uncaged variants of themolecule are imaged by the instant apparatus and methods.

FIG. 1 is an illustration of one embodiment of a cell treatmentapparatus 10. The cell treatment apparatus 10 includes a housing 15 thatstores the inner components of the apparatus. The housing includes lasersafety interlocks to ensure safety of the user, and also limitsinterference by external influences (e.g., ambient light, dust, etc.).Located on the upper portion of the housing 15 is a display unit 20 fordisplaying captured images of cell populations during treatment. Theseimages are captured by a camera, as will be discussed more specificallybelow. A keyboard 25 and mouse 30 are used to input data and control theapparatus 10. An access door 35 provides access to a movable stage thatholds a specimen container of cells undergoing treatment.

An interior view of the apparatus 10 is provided in FIG. 2. Asillustrated, the apparatus 10 provides an upper tray 200 and lower tray210 that hold the interior components of the apparatus. The upper tray200 includes a pair of intake filters 215A,B that filter ambient airbeing drawn into the interior of the apparatus 10. Below the access door35 is the optical subassembly (not shown). The optical subassembly ismounted to the upper tray 200 and is discussed in detail with regard toFIGS. 3-6.

On the lower tray 210 is a computer 225 which stores the softwareprograms, commands and instructions that run the apparatus 10. Inaddition, the computer 225 provides control signals to the treatmentapparatus through electrical signal connections for steering the laserto the appropriate spot on the specimen in order to treat the cells.

As illustrated, a series of power supplies 230A,B,C provide power to thevarious electrical components within the apparatus 10. In addition, anuninterruptable power supply 235 is incorporated to allow the apparatusto continue functioning through short external power interruptions.

FIG. 3 provides a layout of one embodiment of an optical subassemblydesign 300 within an embodiment of a cell treatment apparatus 10. Asillustrated, an illumination laser 305 provides a directed laser outputthat is used to excite a particular label that is attached to targetedcells within the specimen. In this embodiment, the illumination laseremits light at a wavelength of 532 nm. Once the illumination laser hasgenerated a light beam, the light passes into a shutter 310 whichcontrols the pulse length of the laser light.

After the illumination laser light passes through the shutter 310, itenters a ball lens 315 where it is focused into a SMA fiber opticconnector 320. After the illumination laser beam has entered the fiberoptic connector 320, it is transmitted through a fiber optic cable 325to an outlet 330. By passing the illumination beam through the fiberoptic cable 325, the illumination laser 305 can be positioned anywherewithin the treatment apparatus and thus is not limited to only beingpositioned within a direct light pathway to the optical components. Inone embodiment, the fiber optic cable 325 is connected to a vibratingmotor 327 for the purpose of mode scrambling and generating a moreuniform illumination spot.

After the light passes through the outlet 330, it is directed into aseries of condensing lenses in order to focus the beam to the properdiameter for illuminating one frame of cells. As used herein, one frameof cells is defined as the portion of the biological specimen that iscaptured within one frame image captured by the camera. This isdescribed more specifically below.

Accordingly, the illumination laser beam passes through a firstcondenser lens 335. In one embodiment, this first lens has a focallength of 4.6 mm. The light beam then passes through a second condenserlens 340 which, in one embodiment, provides a 100 mm focal length.Finally, the light beam passes into a third condenser lens 345, whichpreferably provides a 200 mm focal length. While the present inventionhas been described using specific condenser lenses, it should beapparent that other similar lens configurations that focus theillumination laser beam to an advantageous diameter would functionsimilarly. Thus, this invention is not limited to the specificimplementation of any particular condenser lens system.

Once the illumination laser beam passes through the third condenser lens345, it enters a cube beamsplitter 350 that is designed to transmit the532 nm wavelength of light emanating from the illumination laser.Preferably, the cube beamsplitter 350 is a 25.4 mm square cube(Melles-Griot, Irvine, Calif.). However, other sizes are anticipated tofunction similarly. In addition, a number of plate beamsplitters orpellicle beamsplitters could be used in place of the cube beamsplitter350 with no appreciable change in function.

Once the illumination laser light has been transmitted through the cubebeamsplitter 350, it reaches a long wave pass mirror 355 that reflectsthe 532 nm illumination laser light to a set of galvanometer mirrors 360that steer the illumination laser light under computer control to ascanning lens (Special Optics, Wharton, N.J.) 365, which directs theillumination laser light to the specimen (not shown). The galvanometermirrors are controlled so that the illumination laser light is directedat the proper cell population (i.e. frame of cells) for imaging. The“scanning lens” described in this embodiment of the invention includes arefractive lens. It should be noted that the term “scanning lens” asused in the present invention includes, but is not limited to, a systemof one or more refractive or reflective optical elements used alone orin combination. Further, the “scanning lens” may include a system of oneor more diffractive elements used in combination with one or morerefractive and/or reflective optical elements. One skilled in the artwill know how to design a “scanning lens” system in order to illuminatethe proper cell population.

The light from the illumination laser is of a wavelength that is usefulfor illuminating the specimen. In this embodiment, energy from acontinuous wave 532 nm Nd:YAG frequency-doubled laser (B&W Tek, Newark,Del.) reflects off the long wave pass mirror (Custom Scientific,Phoenix, Ariz.) and excites fluorescent tags in the specimen. In oneembodiment, the fluorescent tag is phycoerythrin. Alternatively, Alexa532 (Molecular Probes, Eugene, Oreg.) can be used. Phycoerythrin andAlexa 532 have emission spectra with peaks near 580 nm, so that theemitted fluorescent light from the specimen is transmitted via the longwave pass mirror to be directed into the camera. The use of the filterin front of the camera blocks light that is not within the wavelengthrange of interest, thereby reducing the amount of background lightentering the camera.

It is generally known that many other devices could be used in thismanner to illuminate the specimen, including, but not limited to, an arclamp (e.g., mercury, xenon, etc.) with or without filters, alight-emitting diode (LED), other types of lasers, etc. Advantages ofthis particular laser include high intensity, relatively efficient useof energy, compact size, and minimal heat generation. It is alsogenerally known that other fluorochromes with different excitation andemission spectra could be used in such an apparatus with the appropriateselection of illumination source, filters, and long and/or short wavepass mirrors. For example, allophycocyanin (APC) could be excited with a633 nm HeNe illumination laser, and fluoroisothiocyanate (FITC) could beexcited with a 488 nm Argon illumination laser. One skilled in the artcould propose many other optical layouts with various components inorder to achieve the objective of this invention.

In addition to the illumination laser 305, an optional treatment laser400 is present to irradiate the targeted cells once they have beenidentified by image analysis. Of course, in one embodiment, thetreatment induces necrosis of targeted cells within the cell population.As shown, the treatment laser 400 outputs an energy beam of 523 nm thatpasses through a shutter 410. Although the exemplary laser outputs anenergy beam having a 523 nm wavelength, other sources that generateenergy at other wavelengths are also within the scope of the presentinvention.

Once the treatment laser energy beam passes through the shutter 410, itenters a beam expander (Special Optics, Wharton, N.J.) 415 which adjuststhe diameter of the energy beam to an appropriate size at the plane ofthe specimen. Following the beam expander 415 is a half-wave plate 420which controls the polarization of the beam. The treatment laser energybeam is then reflected off a mirror 425 and enters the cube beamsplitter350. The treatment laser energy beam is reflected by 90° in the cubebeamsplitter 350, such that it is aligned with the exit pathway of theillumination laser light beam. Thus, the treatment laser energy beam andthe illumination laser light beam both exit the cube beamsplitter 350along the same light path. From the cube beamsplitter 350, the treatmentlaser beam reflects off the long wave pass mirror 355, is steered by thegalvanometers 360, thereafter contacts the scanning lens 365, andfinally is focused upon a targeted cell within the specimen. Again, the“scanning lens” described in this embodiment includes a refractive lens.As previously mentioned, the term “scanning lens” includes, but is notlimited to, a system of one or more refractive or reflective opticalelements used alone or in combination. Further, the “scanning lens” mayinclude one or more diffractive elements used in combination with one ormore refractive and/or reflective elements. One skilled in the art willknow how to design a “scanning lens” system in order to focus upon thetargeted cell within the specimen

It should be noted that a small fraction of the illumination laser lightbeam passes through the long wave pass mirror 355 and enters a powermeter sensor (Gentec, Palo Alto, Calif.) 445. The fraction of the beamentering the power sensor 445 is used to calculate the level of poweremanating from the illumination laser 305. In an analogous fashion, asmall fraction of the treatment laser energy beam passes through thecube beamsplitter 350 and enters a second power meter sensor (Gentec,Palo Alto, Calif.) 446. The fraction of the beam entering the powersensor 446 is used to calculate the level of power emanating from thetreatment laser 400. The power meter sensors are electrically linked tothe computer system so that instructions/commands within the computersystem capture the power measurement and determine the amount of energythat was emitted.

The energy beam from the treatment laser is of a wavelength that isuseful for achieving a response in the cells. In the example shown, apulsed 523 nm Nd:YLF frequency-doubled laser is used to heat a localizedvolume containing the targeted cell, such that it is induced to diewithin a pre-determined period of time. The mechanism of death isdependent upon the actual temperature achieved in the cell, as reviewedby Niemz (Niemz 1996).

A Nd:YLF frequency-doubled, solid-state laser (Spectra-Physics, MountainView, Calif.) is used because of its stability, high repetition rate offiring, and long time of maintenance-free service. However, most cellculture fluids and cells are relatively transparent to light in thisgreen wavelength, and therefore a very high fluence of energy would berequired to achieve cell death. To significantly reduce the amount ofenergy required, and therefore the cost and size of the treatment laser,a dye is purposefully added to the specimen to efficiently absorb theenergy of the treatment laser in the specimen. In the example shown, thenon-toxic dye FD&C red #40 (allura red) is used to absorb the 523 nmenergy from the treatment laser, but one skilled in the art couldidentify other laser/dye combinations that would result in efficientabsorption of energy by the specimen. For example, a 633 nm HeNe laser'senergy would be efficiently absorbed by FD&C green #3 (fast green FCF),a 488 nm Argon laser's energy would be efficiently absorbed by FD&Cyellow #5 (sunset yellow FCF), and a 1064 nm Nd:YAG laser's energy wouldbe efficiently absorbed by Filtron (Gentex, Zeeland, Mich.) infraredabsorbing dye. Through the use of an energy absorbing dye, the amount ofenergy required to kill a targeted cell can be reduced since more of thetreatment laser energy is absorbed in the presence of such a dye.

Another method of achieving thermal killing of cells without theaddition of a dye involves the use of an ultraviolet laser. Energy froma 355 nm Nd:YAG frequency-tripled laser will be absorbed by nucleicacids and proteins within the cell, resulting in thermal heating anddeath. Yet another method of achieving thermal killing of cells withoutthe addition of a dye involves the use of a near-infrared laser. Energyfrom a 2100 nm Ho:YAG laser or a 2940 nm Er:YAG laser will be absorbedby water within the cell, resulting in thermal heating and death.

Although this embodiment describes the killing of cells via thermalheating by the energy beam, one skilled in the art would recognize thatother responses can also be induced in the cells by an energy beam,including photomechanical disruption, photodissociation, photoablation,and photochemical reactions, as reviewed by Niemz (Niemz 1996). Forexample, a photosensitive substance (e.g., hemtoporphyrin derivative,tin-etiopurpurin, lutetuim texaphyrin) (Oleinick and Evans 1998) withinthe cell mixture could be specifically activated in targeted cells byirradiation. Additionally, a small, transient pore could be made in thecell membrane (Palumbo et al. 1996) to allow the entry of genetic orother material. Further, specific molecules in or on the cell, such asproteins or genetic material, could be inactivated by the directedenergy beam (Grate and Wilson 1999; Jay 1988). Also, photobleaching canbe utilized to measure intracellular movements such as the diffusion ofproteins in membranes and the movements of microtubules during mitosis(Ladha et al., 1997 J. Cell Sci 1997 110(9): 1041; Centonze and Borisy,1991 J. Cell Sci 100 (pt 1):205; White and Stelzer, Trends Cell Biol1999 February; 9(2):61-5; Meyvis, et al., Pharm Res 1999 August; 16(8):1153-62). Further, photolysis or uncaging, including multiphotonuncaging, of caged compounds can be utilized to control the release,with temporal and spacial resolution, of biologically active products orother products of interest (Theriot and Mitchison, 1992 J. Cell Biol.119:367; Denk, 1994 PNAS 91(14):6629). These mechanisms of inducing aresponse in a targeted cell via the use of electromagnetic radiationdirected at specific targeted cells are also intended to be incorporatedinto the present invention.

In addition to the illumination laser 305 and treatment laser 400, theapparatus includes a camera 450 that captures images (i.e. frames) ofthe cell populations. As illustrated in FIG. 3, the camera 450 isfocused through a lens 455 and filter 460 in order to accurately recordan image of the cells without capturing stray background images. A stop462 is positioned between the filter 460 and mirror 355 in order toeliminate light that may enter the camera from angles not associatedwith the image from the specimen. The filter 460 is chosen to only allowpassage of light within a certain wavelength range. This wavelengthrange includes light that is emitted from the targeted cells uponexcitation by the illumination laser 305, as well as light from aback-light source 475.

The back-light source 475 is located above the specimen to provideback-illumination of the specimen at a wavelength different from thatprovided by the illumination laser 305. This LED generates light at 590nm, such that it can be transmitted through the long wave pass mirror tobe directed into the camera. This back-illumination is useful forimaging cells when there are no fluorescent targets within the framebeing imaged. An example of the utility of this back-light is its use inattaining proper focus of the system, even when there are onlyunstained, non-fluorescent cells in the frame. In one embodiment, theback-light is mounted on the underside of the access door 35 (FIG. 2).

Thus, as discussed above, the only light returned to the camera is fromwavelengths that are of interest in the specimen. Other wavelengths oflight do not pass through the filter 460, and thus do not becomerecorded by the camera 450. This provides a more reliable mechanism forcapturing images of only those cells of interest. It is readily apparentto one skilled in the art that the single filter 460 could be replacedby a movable filter wheel that would allow different filters to be movedin and out of the optical pathway. In such an embodiment, images ofdifferent wavelengths of light could be captured at different timesduring cell processing, allowing the use of multiple cell labels.

It should be noted that in this embodiment, the camera is acharge-coupled device (CCD) and transmits images back to the computersystem for processing. As will be described below, the computer systemdetermines the coordinates of the targeted cells in the specimen byreference to the image captured by the CCD camera.

Referring now to FIG. 4, a perspective view of an embodiment of anoptical subassembly is illustrated. As illustrated, the illuminationlaser 305 sends a light beam through the shutter 310 and ball lens 315to the SMA fiber optic connector 320. The light passes through the fiberoptic cable 325 and through the output 330 into the condenser lenses335, 340 and 345. The light then enters the cube beamsplitter 350 and istransmitted to the long wave pass mirror 355. From the long wave passmirror 355, the light beam enters the computer-controlled galvanometers360 and is then steered to the proper frame of cells in the specimenfrom the scanning lens 365.

As also illustrated in the perspective drawing of FIG. 4, the treatmentlaser 400 transmits energy through the shutter 410 and into the beamexpander 415. Energy from the treatment laser 400 passes through thebeam expander 415 and passes through the half-wave plate 420 beforehitting the fold mirror 425, entering the cube beamsplitter 350 where itis reflected 90° to the long wave pass mirror 355, from which it isreflected into the computer controlled galvanometer mirrors 360. Afterbeing steered by the galvanometer mirrors 360 to the scanning lens 365,the laser energy beam strikes the proper location within the cellpopulation in order to induce a response in a particular targeted cell.

In order to accommodate a very large surface area of specimen to treat,the apparatus includes a movable stage that mechanically moves thespecimen container with respect to the scanning lens. Thus, once aspecific sub-population (i.e. field) of cells within the scanning lensfield-of-view has been treated, the movable stage brings anothersub-population of cells within the scanning lens field-of-view. Asillustrated in FIG. 5, a computer-controlled movable stage 500 holds aspecimen container (not shown) to be processed. The movable stage 500 ismoved by computer-controlled servo motors along two axes so that thespecimen container can be moved relative to the optical components ofthe instrument. The stage movement along a defined path is coordinatedwith other operations of the apparatus. In addition, specificcoordinates can be saved and recalled to allow return of the movablestage to positions of interest. Encoders on the x and y movement provideclosed-loop feedback control on stage position.

The flat-field (F-theta) scanning lens 365 is mounted below the movablestage. The scanning lens field-of-view comprises the portion of thespecimen that is presently positioned above the scanning lens by themovable stage 500. The lens 365 is mounted to a stepper motor thatallows the lens 365 to be automatically raised and lowered (along thez-axis) for the purpose of focusing the system.

As illustrated in FIGS. 4-6, below the scanning lens 365 are thegalvanometer-controlled steering mirrors 360 that deflectelectromagnetic energy along two perpendicular axes. Behind the steeringmirrors is the long wave pass mirror 355 that reflects electromagneticenergy of a wavelength shorter than 545 nm. Wavelengths longer than 545nm are passed through the long wave pass mirror, directed through thefilter 460, coupling lens 455, and into the CCD camera, therebyproducing an image of the appropriate size on the CCD sensor of thecamera 450 (See FIGS. 3 and 4). The magnification defined by thecombination of the scanning lens 365 and coupling lens 455 is chosen toreliably detect single cells while maximizing the area viewed in oneframe by the camera. Although a CCD camera (DVC, Austin, Tex.) isillustrated in this embodiment, the camera can be any type of detectoror image gathering equipment known to those skilled in the art. Theoptical subassembly of the apparatus is preferably mounted on avibration-isolated platform to provide stability during operation asillustrated in FIGS. 2 and 5.

Referring now to FIG. 7, a top view of the movable stage 500 isillustrated. As shown, a specimen container is mounted in the movablestage 500. The specimen container 505 rests on an upper axis nest plate510 that is designed to move in the forward/backward direction withrespect to the movable stage 500. A stepper motor (not shown) isconnected to the upper axis nest plate 510 and computer system so thatcommands from the computer cause forward/backward movement of thespecimen container 505.

The movable stage 500 is also connected to a timing belt 515 thatprovides side-to-side movement of the movable stage 500 along a pair ofbearing tracks 525A,B. The timing belt 515 attaches to a pulley (notshown) housed under a pulley cover 530. The pulley is connected to astepper motor 535 that drives the timing belt 515 to result inside-to-side movement of the movable stage 500. The stepper motor 535 iselectrically connected to the computer system so that commands withinthe computer system result in side-to-side movement of the movable stage500. A travel limit sensor 540 connects to the computer system andcauses an alert if the movable stage travels beyond a predeterminedlateral distance.

A pair of accelerometers 545A,B is preferably incorporated on thisplatform to register any excessive bumps or vibrations that mayinterfere with the apparatus operation. In addition, a two-axisinclinometer 550 is preferably incorporated on the movable stage toensure that the specimen container is level, thereby reducing thepossibility of gravity-induced motion in the specimen container.

The specimen chamber has a fan with ductwork to eliminate condensationon the specimen container, and a thermocouple to determine whether thespecimen chamber is within an acceptable temperature range. Additionalfans are provided to expel the heat generated by the electroniccomponents, and appropriate filters are used on the air intakes 215A,B.

The computer system 225 controls the operation and synchronization ofthe various pieces of electronic hardware described above. The computersystem can be any commercially available computer that can interfacewith the hardware. One example of such a computer system is an IntelPentium II, III or IV-based computer running the Microsoft Windows® NToperating system. Software is used to communicate with the variousdevices, and control the operation in the manner that is describedbelow.

When the apparatus is first initialized, the computer loads files fromthe hard drive into RAM for proper initialization of the apparatus. Anumber of built-in tests are automatically performed to ensure theapparatus is operating properly, and calibration routines are executedto calibrate the apparatus. Upon successful completion of theseroutines, the user is prompted to enter information via the keyboard andmouse regarding the procedure that is to be performed. Once the requiredinformation is entered, the user is prompted to open the access door 35and load a specimen onto the movable stage.

Once a specimen is in place on the movable stage and the door is closed,the computer passes a signal to the stage to move into a home position.The fan is initialized to begin warming and defogging of the specimen.During this time, cells within the specimen are allowed to settle to thebottom surface. In addition, during this time, the apparatus may runcommands that ensure that the specimen is properly loaded, and is withinthe focal range of the system optics. For example, specific markings onthe specimen container can be located and focused on by the system toensure that the scanning lens has been properly focused on the bottom ofthe specimen container. Such markings could also be used by theinstrument to identify the container, its contents, and even theprocedure to be performed. After a suitable time, the computer turns offthe fan to prevent excess vibrations during treatment, and cellprocessing begins.

First, the computer instructs the movable stage to be positioned overthe scanning lens so that the first area (i.e. field) of the specimen tobe treated is directly in the scanning lens field-of-view. Thegalvanometer mirrors are instructed to move such that the center framewithin the field-of-view is imaged in the camera. As discussed below,the field imaged by the scanning lens is separated into a plurality offrames. Each frame is the proper size so that the cells within the frameare effectively imaged by the camera.

The back-light 475 is then activated in order to illuminate thefield-of-view so that it can be brought into focus by the scanning lens.Once the scanning lens has been properly focused upon the specimen, thecomputer system divides the field-of-view into a plurality of frames sothat each frame is analyzed separately by the camera. This methodologyallows the apparatus to process a plurality of frames within a largefield-of-view without moving the mechanical stage. Because thegalvanometers can move from one frame to the next very rapidly comparedto the mechanical steps involved in moving the stage, this methodresults is an extremely fast and efficient apparatus.

Other means of ensuring that the specimen is in focus are alsoavailable. For example, a laser proximeter (Cooke Corp., Auburn, Mich.)could rapidly determine the distance between the scanning lens and thesample, and adjust the scanning lens position accordingly. Ultrasonicproximeters are also available, and would achieve the same objective.One skilled in the art could propose other means of ensuring that thespecimen is in focus above the scanning lens.

In one preferred embodiment, the apparatus described herein processes atleast 1, 2, 3, 4, 5, 6, 7, or 14 square centimeters of a biologicalspecimen per minute. In another embodiment, the apparatus describedherein processes at least 0.25, 0.5, 1, 2, 3, 4 or 8 million cells of abiological specimen per minute. In one other embodiment, the apparatuscan preferably induce a response in targeted cells at a rate of 50, 100,150, 200, 250, 300, 350, 400 or 800 cells per second.

Initially, an image of the frame at the center of the field-of-view iscaptured by the camera and stored to a memory in the computer.Instructions in the computer analyze the focus of the specimen bylooking at the size of, number of, and other object features in theimage. If necessary, the computer instructs the z-axis motor attached tothe scanning lens to raise or lower in order to achieve the best focus.The apparatus may iteratively analyze the image at several z-positionsuntil the best focus is achieved. The galvanometer-controlled mirrorsare then instructed to image a first frame, within the field-of-view, inthe camera. For example, the entire field-of-view might be divided into4, 9, 12, 18, 24 or more separate frames that will be individuallycaptured by the camera. Once the galvanometer mirrors are pointed to thefirst frame in the field-of-view, the shutter in front of theillumination laser is opened to illuminate the first frame through thegalvanometer mirrors and scanning lens. The camera captures an image ofany fluorescent emission from the specimen in the first frame of cells.Once the image has been acquired, the shutter in front of theillumination laser is closed and a software program (Epic, BuffaloGrove, Ill.) within the computer processes the image.

The power sensor 445 discussed above detects the level of light that wasemitted by the illumination laser, thereby allowing the computer tocalculate if it was adequate to illuminate the frame of cells. If not,another illumination and image capture sequence is performed. Repeatedfailure to sufficiently illuminate the specimen will result in an errorcondition that is communicated to the operator.

Shuttering of illumination light reduces undesirable heating andphotobleaching of the specimen and provides a more repeatablefluorescent signal. An image analysis algorithm is run to locate the x-ycentroid coordinates of all targeted cells in the frame by reference tofeatures in the captured image. If there are targets in the image, thecomputer calculates the two-dimensional coordinates of all targetlocations in relation to the movable stage position and field-of-view,and then positions the galvanometer-controlled mirrors to point to thelocation of the first target in the first frame of cells. It should benoted that only a single frame of cells within the field-of-view hasbeen captured and analyzed at this point. Thus, there should be arelatively small number of identified targets within this sub-populationof the specimen. Moreover, because the camera is pointed to a smallerpopulation of cells, a higher magnification is used so that each targetis imaged by many pixels within the CCD camera.

Once the computer system has positioned the galvanometer controlledmirrors to point to the location of the first targeted cell within thefirst frame of cells, the treatment laser is fired for a brief intervalso that the first targeted cell is given an appropriate dose of energy.The power sensor 446 discussed above detects the level of energy thatwas emitted by the treatment laser, thereby allowing the computer tocalculate if it was adequate to induce a response in the targeted cell.If not sufficient, the treatment laser is fired at the same targetagain. If repeated shots do not deliver the required energy dose, anerror condition is communicated to the operator. These targeting,firing, and sensing steps are repeated by the computer for all targetsidentified in the captured frame.

Once all of the targets have been irradiated with the treatment laser inthe first frame of cells, the mirrors are then positioned to the secondframe of cells in the field-of-view, and the processing repeats at thepoint of frame illumination and camera imaging. This processingcontinues for all frames within the field-of-view above the scanninglens. When all of these frames have been processed, the computerinstructs the movable stage to move to the next field-of-view in thespecimen, and the process repeats at the back-light illumination andauto-focus step. Frames and fields-of-view are appropriately overlappedto reduce the possibility of inadvertently missing areas of thespecimen. Once the specimen has been fully processed, the operator issignaled to remove the specimen, and the apparatus is immediately readyfor the next specimen.

Although the text above describes the analysis of fluorescent images forlocating targets, one can easily imagine that the non-fluorescentback-light LED illumination images will be useful for locating othertypes of targets as well, even if they are unlabeled.

The advantage of using the galvanometer mirrors to control the imagingof successive frames and the irradiation of successive targets issignificant. One brand of galvanometer is the Cambridge Technology, Inc.model number 6860 (Cambridge, Mass.). This galvanometer can repositionvery accurately within a few milliseconds, making the processing oflarge areas and many targets possible within a reasonable amount oftime. In contrast, the movable stage is relatively slow, and istherefore used only to move specified areas of the specimen into thescanning lens field-of-view. Error signals continuously generated by thegalvanometer control boards are monitored by the computer to ensure thatthe mirrors are in position and stable before an image is captured, orbefore a target is fired upon, in a closed-loop fashion.

In the context of the present invention, the term “specimen” has a broadmeaning. It is intended to encompass any type of biological sampleplaced within the apparatus. The specimen may be enclosed by, orassociated with, a container to maintain the sterility and viability ofthe cells. Further, the specimen may incorporate, or be associated with,a cooling apparatus to keep it above or below ambient temperature duringoperation of the methods described herein. The specimen container, ifone is used, must be compatible with the use of the illumination laser,back-light illuminator, and treatment laser, such that it transmitsadequate energy without being substantially damaged itself.

Of course, many variations of the above-described embodiment arepossible, including alternative methods for illuminating, imaging, andtargeting the cells. For example, movement of the specimen relative tothe scanning lens could be achieved by keeping the specimensubstantially stationary while the scanning lens is moved. Steering ofthe illumination beam, images, and energy beam could be achieved throughany controllable reflective or diffractive device, including prisms,piezo-electric tilt platforms, or acousto-optic deflectors.Additionally, the apparatus can image/process from either below or abovethe specimen. Because the apparatus is focused through a movablescanning lens, the illumination and energy beams can be directed todifferent focal planes along the z-axis. Thus, portions of the specimenthat are located at different vertical heights can be specificallyimaged and processed by the apparatus in a three-dimensional manner. Thesequence of the steps could also be altered without changing theprocess. For example, one might locate and store the coordinates of alltargets in the specimen, and then return to the targets to irradiatethem with energy one or more times over a period of time.

To optimally process the specimen, it should be placed on asubstantially flat surface so that a large portion of the specimenappears within a narrow range of focus, thereby reducing the need forrepeated auto-focus steps. The density of cells on this surface can, inprinciple, be at any value. However, the cell density should be as highas possible to minimize the total surface area required for theprocedure.

The following examples illustrate the use of the described method andapparatus in different applications.

Example 1 Autologous HSC Transplantation

A patient with a B cell-derived metastatic tumor in need of anautologous HSC transplant is identified by a physician. As a first stepin the treatment, the patient undergoes a standard HSC harvestprocedure, resulting in collection of approximately 1×10¹⁰ hematopoieticcells with an unknown number of contaminating tumor cells. The harvestedcells are enriched for HSC by a commercial immunoaffinity column(Isolex® 300, Nexell Therapeutics, Irvine, Calif.) that selects forcells bearing the CD34 surface antigen, resulting in a population ofapproximately 3×10⁸ hematopoietic cells, with an unknown number of tumorcells. The mixed population is thereafter contacted with anti-B cellantibodies (directed against CD20 and CD22) that are conjugated tophycoerythrin. The labeled antibodies specifically bind to the Bcell-derived tumor cells.

The mixed cell population is then placed in a sterile specimen containeron a substantially flat surface near confluence, at approximately500,000 cells per square centimeter. The specimen is placed on themovable stage of the apparatus described above, and all detectable tumorcells are identified by reference to phycoerythrin and targeted with alethal dose of energy from a treatment laser. The design of theapparatus allows the processing of a clinical-scale transplant specimenin under 4 hours. The cells are recovered from the specimen container,washed, and then cryopreserved. Before the cells are reinfused, thepatient is given high-dose chemotherapy to destroy the tumor cells inthe patient's body. Following this treatment, the processed cells arethawed at 37° C. and are given to the patient intravenously. The patientsubsequently recovers with no remission of the original cancer.

Example 2 Allogeneic HSC Transplantation

In another embodiment, the significant risk and severity ofgraft-versus-host disease in the allogeneic HSC transplant setting canbe combated. A patient is selected for an allogeneic transplant once asuitable donor is found. Cells are harvested from the selected donor asdescribed in the above example. In this case, the cell mixture iscontacted with phycoerythrin-labeled anti-CD3 T-cell antibodies.Alternatively, specific allo-reactive T-cell subsets could be labeledusing an activated T-cell marker (e.g. CD69) in the presence ofallo-antigen. The cell population is processed by the apparatusdescribed herein, thereby precisely defining and controlling the numberof T-cells given to the patient. This type of control is advantageous,because administration of too many T-cells increases the risk ofgraft-versus-host disease, whereas too few T-cells increases the risk ofgraft failure and the risk of losing of the known beneficialgraft-versus-leukemia effect. The present invention and methods arecapable of precisely controlling the number of T-cells in an allogeneictransplant.

Example 3 Tissue Engineering

In another application, the present apparatus is used to removecontaminating cells in inocula for tissue engineering applications. Cellcontamination problems exist in the establishment of primary cellcultures required for implementation of tissue engineering applications,as described by Langer and Vacanti (Langer and Vacanti 1999). Inparticular, chondrocyte therapies for cartilage defects are hampered byimpurities in the cell populations derived from cartilage biopsies.Accordingly, the present invention is used to specifically remove thesetypes of cells from the inocula.

For example, a cartilage biopsy is taken from a patient in need ofcartilage replacement. The specimen is then grown under conventionalconditions (Brittberg et al. 1994). The culture is then stained with aspecific label for any contaminating cells, such as fast-growingfibroblasts. The cell mixture is then placed within the apparatusdescribed and the labeled, contaminating cells are targeted by thetreatment laser, thereby allowing the slower growing chondrocytes tofully develop in culture.

Example 4 Stem Cell Therapy

Yet another embodiment involves the use of embryonic stem cells to treata wide variety of diseases. Since embryonic stem cells areundifferentiated, they can be used to generate many types of tissue thatwould find use in transplantation, such as cardiomyocytes and neurons.However, undifferentiated embryonic stem cells that are implanted canalso lead to a jumble of cell types which form a type of tumor known asa teratoma (Pedersen 1999). Therefore, therapeutic use of tissuesderived from embryonic stem cells must include rigorous purification ofcells to ensure that only sufficiently differentiated cells areimplanted. The apparatus described herein is used to eliminateundifferentiated stem cells prior to implantation of embryonic stemcell-derived tissue in the patient.

Example 5 Generation of Human Tumor Cell Cultures

In another embodiment, a tumor biopsy is removed from a cancer patientfor the purpose of initiating a culture of human tumor cells. However,the in vitro establishment of primary human tumor cell cultures frommany tumor types is complicated by the presence of contaminating primarycell populations that have superior in vitro growth characteristics overtumor cells. For example, contaminating fibroblasts represent a majorchallenge in establishing many cancer cell cultures. The disclosedapparatus is used to particularly label and destroy the contaminatingcells, while leaving the biopsied tumor cells intact. Accordingly, themore aggressive primary cells will not overtake and destroy the cancercell line.

Example 6 Generation of a Specific mRNA Expression Library

The specific expression pattern of genes within different cellpopulations is of great interest to many researchers, and many studieshave been performed to isolate and create libraries of expressed genesfor different cell types. For example, knowing which genes are expressedin tumor cells versus normal cells is of great potential value (Cossmanet al. 1999). Due to the amplification methods used to generate suchlibraries (e.g. PCR), even a small number of contaminating cells willresult in an inaccurate expression library (Cossman et al. 1999; Schutzeand Lahr 1998). One approach to overcome this problem is the use oflaser capture microdissection (LCM), in which a single cell is used toprovide the starting genetic material for amplification (Schutze, Lahr1998). Unfortunately, gene expression in single cells is somewhatstochastic, and may be biased by the specific state of that individualcell at the time of analysis (Cossman et al. 1999). Therefore, accuratepurification of a significant cell number prior to extraction of mRNAwould enable the generation of a highly accurate expression library, onethat is representative of the cell population being studied, withoutbiases due to single cell expression or expression by contaminatingcells. The methods and apparatus described in this invention can be usedto purify cell populations so that no contaminating cells are presentduring an RNA extraction procedure.

Example 7 Transfection of a Specific Cell Population

Many research and clinical gene therapy applications are hampered by theinability to transfect an adequate number of a desired cell type withouttransfecting other cells that are present. The method of the presentinvention would allow selective targeting of cells to be transfectedwithin a mixture of cells. By generating a photomechanical shock wave ator near a cell membrane with a targeted energy source, a transient porecan be formed, through which genetic (or other) material can enter thecell. This method of gene transfer has been called optoporation (Palumboet al. 1996). The apparatus described above can achieve selectiveoptoporation on only the cells of interest in a rapid, automated,targeted manner.

For example, white blood cells are plated in a specimen container havinga solution containing DNA to be transfected. Fluorescently-labeledantibodies having specificity for stem cells are added into the mediumand bind to the stem cells. The specimen container is placed within thecell processing apparatus and a treatment laser is targeted to any cellsthat become fluorescent under the illumination laser light. Thetreatment laser facilitates transfection of DNA specifically into thetargeted cells.

Example 8 Selection of Desirable Clones in a Biotechnology Application

In many biotechnology processes where cell lines are used to generate avaluable product, it is desirable to derive clones that are veryefficient in producing the product. This selection of clones is oftencarried out manually, by inspecting a large number of clones that havebeen isolated in some manner. The present invention would allow rapid,automated inspection and selection of desirable clones for production ofa particular product. For example, hybridoma cells that are producingthe greatest amounts of antibody can be identified by a fluorescentlabel directed against the F_(c) region. Cells with no or dimfluorescent labeling are targeted by the treatment laser for killing,leaving behind the best producing clones for use in antibody production.

Example 9 Automated Monitoring of Cellular Responses

Automated monitoring of cellular responses to specific stimuli is ofgreat interest in high-throughput drug screening. Often, a cellpopulation in one well of a well-plate is exposed to a stimulus, and afluorescent signal is then captured over time from the cell populationas a whole. Using the methods and apparatus described herein, moredetailed monitoring could be done at the single cell level. For example,a cell population can be labeled to identify a characteristic of asubpopulation of cells that are of interest. This label is then excitedby the illumination laser to identify those cells. Thereafter, thetreatment laser is targeted at the individual cells identified by thefirst label, for the purpose of exciting a second label, therebyproviding information about each cell's response. Since the cells aresubstantially stationary on a surface, each cell could be evaluatedmultiple times, thereby providing temporal information about thekinetics of each cell's response. Also, through the use of the largearea scanning lens and galvanometer mirrors, a relatively large numberof wells could be quickly monitored over a short period of time.

As a specific example, consider the case of alloreactive T-cells aspresented in Example 2 above. In the presence of allo-antigen, activateddonor T-cells could be identified by CD69. Instead of using thetreatment laser to target and kill these cells, the treatment lasercould be used to examine the intracellular pH of every activated T-cellthrough the excitation and emitted fluorescence of carboxyfluoresceindiacetate. The targeted laser allows the examination of only cells thatare activated, whereas most screening methods evaluate the response ofan entire cell population. If a series of such wells are being monitoredin parallel, various agents could be added to individual wells, and thespecific activated T-cell response to each agent could be monitored overtime. Such an apparatus would provide a high-throughput screening methodfor agents that ameliorate the alloreactive T-cell response ingraft-versus-host disease. Based on this example, one skilled in the artcould imagine many other examples in which a cellular response to astimulus is monitored on an individual cell basis, focusing only oncells of interest identified by the first label.

Example 10 Photobleaching Studies

Photobleaching, and/or photobleach recovery, of a specific area of afluorescently-stained biological sample is a common method that is usedto assess various biological processes. For example, a cell suspensionis labeled with rhodamine 123, which fluorescently stains mitochondriawithin the cells. Using the instant illumination laser, the mitochondriawithin one or more cells are visualized due to rhodamine 123fluorescence. The treatment laser is then used to deliver a focused beamof light that results in photobleaching of the rhodamine 123 in a smallarea within one or more cells. The photobleached area(s) then appeardark immediately thereafter, whereas adjacent areas are unaffected. Aseries of images are then taken using the illumination laser, providinga time-lapse series of images that document the migration of unbleachedmitochondria into the area that was photobleached with the treatmentlaser. This approach can be used to assess the motion, turnover, orreplenishment of many biological structures within cells.

Thus, in cultured rat neurites, the photobleach recovery of mitochondriais a measure of the size of the mobile pool of mitochondria within eachcell (Chute, et al. 1995). The rate of photobleach recovery in thesecells is dependent on intracellular calcium and magnesiumconcentrations, energy status, and microtubule integrity. Neurotoxicsubstances, such as taxol or vinblastine, will affect the rate ofphotobleach recovery. Therefore, an assay for neurotoxic substancescould be based on the measurement of photobleach recovery ofmitochondria within a statistically significant number of neurites thathad been exposed to various agents in the wells of a multi-well plate.In such an application, the apparatus described herein and used asdescribed above, would provide a rapid automated method to assessneurotoxicity of many substances on a large number of cells. Based onthis example, one skilled in the art could imagine many other examplesin which photobleaching is induced and photobleach recovery is monitoredin order to obtain useful information from a biological specimen.

Example 11 Uncaging Studies

Use of caged compounds to study rapid biological processes involves thebinding (i.e. caging) of a biologically relevant substance in aninactive state, allowing the caged substance to diffuse into thebiological specimen (a relatively slow process), and then using a laserto induce a photolysis reaction (a relatively fast process) whichliberates (i.e. uncages) the substance in situ over microsecond timescales. The biological specimen is then observed in short time-lapsemicroscopy in order to determine the effect of the uncaged substance onsome biological process. Cages for many important substances have beendescribed, including Dioxygen, cyclic ADP-ribose (cADPR), nicotinic acidadenine dinucleotide phosphate (NAADP), nitric oxide (NO), calcium,L-aspartate, and adenosine triphosphate (ATP). Chemotaxis is one exampleof a physiological characteristic that can be studied by uncagingcompounds.

Uncaging studies involve the irradiation of a portion of a biologicalspecimen with laser light followed by examination of the specimen withtime-lapse microscopy. The apparatus of the current invention has clearutility in such studies. As a specific example, consider the study of E.coli chemotaxis towards L-aspartate (Jasuja, et al. 1999). Thebeta-2,6-dinitrobenzyl ester of L-aspartic acid and the1-(2-nitrophenyl)ethyl ether of 8-hydroxypyrene-1,3,6-tris-sulfonic acidare added to the wells of a well plate containing E. coli. Uponirradiation with the treatment laser, a localized uncaging ofL-aspartate and the fluorophore 8-hydroxypyrene-1,3,6-tris-sulfonic acid(pyranine) is induced. The L-aspartate acts as a chemoattractant for E.coli, and in subsequent fluorescent images (using the illuminationlaser) the pyranine fluorophore acts as an indicator of the degree ofuncaging that has occurred in the local area of irradiation. Time-lapseimages of the E. coli. in the vicinity illuminated by visible wavelengthlight, such as from the back-light, of the uncaging event are used tomeasure the chemotactic response of the microorganisms to the locallyuncaged L-aspartate. Due to the nature of the present invention, a largenumber of wells, each with a potential anti-microbial agent added, arescreened in rapid order to determine the chemotactic response ofmicroorganisms. Based on this example, one skilled in the art couldimagine many other examples in which uncaging is induced by thetreatment laser, followed by time-lapse microscopy in order to obtainuseful information on a large number of samples in an automated fashion.

Although aspects of the present invention have been described byparticular embodiments exemplified herein, the present invention is notso limited. The present invention is only limited by the claims appendedbelow.

REFERENCES CITED

U.S. Patent Documents 4,284,897 Sawamura et al. 4,395,397 Shapiro4,532,402 Overbeck 4,629,687 Schindler, et al. 5,035,693 Kratzer5,296,963 Murakami et al. 5,381,224 Dixon et al. 5,646,411 Kain5,672,880 Kain 5,690,846 Okada et al. 5,719,391 Kain 5,932,872 Price

Other Publications

-   Andersen, N. S., Donovan, J. W., Borus, J. S., Poor, C. M., Neuberg,    D., Aster, J. C., Nadler, L. M., Freedman, A. S., and Gribben, J.    G.: Failure of immunologic purging in mantle cell lymphoma assessed    by polymerase chain reaction detection in minimal residual disease.    Blood 90: 4212-4221, 1997-   Brittberg, M., Lindahl, A., Nilsson, A., Ohlsson, C., Isaksson, O.,    and Peterson, L.: Treatment of deep cartilage defects in the knee    with autologous chondrocyte transplantation. N.E.J. Med. 331:    889-895, 1994-   Brockstein, B. E., Ross, A. A., Moss, T. J., Kahn, D. G.,    Hollingsworth, K., and Williams, S. F.: Tumor cell contamination of    bone marrow harvest products: Clinical consequences in a cohort of    advanced-stage breast cancer patients undergoing high-dose    chemotherapy. J. Hematotherapy 5: 617-624, 1996-   Chute, S. K., Flint O. P., and Durham S. K. (1995 Analysis of the    steady-state dynamics organelle motion in cultured neurites: Clin    Exp Pharmco Physiol 22:360.-   Cossman, J. C., Annunziata, C. M., Barash, S., Staudt, L., Dillon,    P., He, W.-W., Ricciardi-Castognoli, P., Rosen, C. A., and    Carter, K. C.: Reed-Sternberg cell genome expression supports a    B-cell lineage. Blood 94: 411-416, 1999-   Deisseroth, A. B., Zu, Z., Claxton, D., Hanania, E. G., Fu, S.,    Ellerson, D., Goldberg, L., Thomas, M., Janicek, K., Anderson, W.    F., Hester, J., Korbling, M., Durett, A., Moen, R., Berenson, R.,    Heimfeld, S., Hamer, J., Calver, L., Tibbits, P., Talpaz, M.,    Kantarjiam, H., Champlin, R., and Reading, C.: Genetic marking shows    that Ph⁺ cells present in autologous transplants of chronic    myelogenous leukemia (CML) contribute to relapse after autologous    bone marrow transplantation in CML. Blood 83: 3068-3076, 1994-   Fields, K. K., Elfenbein, G. J., Trudeau, W. L., Perkins, J. B.,    Janssen, W. E., and Moscinski, L. C.: Clinical significance of bone    marrow metastases as detected using the polymerase chain reaction in    patients with breast cancer undergoing high-dose chemotherapy and    autologous bone marrow transplantation. J. Clin. Oncol. 14:    1868-1876, 1996-   Gazitt, Y., Reading, C. C., Hoffman, R., Wickrema, A., Vesole, D.    H., Jagannath, S., Condino, J., Lee, B., Barlogie, B., and Tricot,    G.: Purified CD34⁺lin-Thy⁺ stem cells do not contain clonal myeloma    cells. Blood 86: 381-389, 1995-   Grate, D. and Wilson, C.: Laser-mediated, site-specific inactivation    of RNA transcripts. PNAS 96: 6131-6136, 1999-   Gribben, J. G., Freedman, A. S., Neuberg, D., Roy, D. C., Blake, K.    W., Woo, S. D., Grossbard, M. L., Rabinowe, S, N., Coral, F.,    Freeman, G. J., Ritz, J., and Nadler, L. M.: Immunologic purging of    marrow assessed by PCR before autologous bone marrow transplantation    for B-cell lymphoma. N.E.J. Med. 325: 1525-1533, 1991-   Jasuja R., Keyoung J., Reid G. P., Trentham D. R., Khan S:    Chemotactic responses of Escherichia coli to small jumps of    photoreleased L-aspartate. Giophys J. 76:1706 (1999).-   Jay, D. G.: Selective destruction of protein function by    chromophore-assisted laser inactivation. PNAS 85: 5454-5458, 1988-   Langer, R. S, and Vacanti, J. P.: Tissue engineering: The challenges    ahead. Sci. Am. 280: 86-89, 1999-   Mapara, M. Y., Körner, I. J., Hildebrandt, M., Bargou, R., Krahl,    D., Reichardt, P., and Dorken, B.: Monitoring of tumor cell purging    after highly efficient immunomagnetic selection of CD34 cells from    leukapheresis products in breast cancer patients: Comparison of    immunocytochemical tumor cell staining and reverse    transcriptase-polymerase chain reaction. Blood 89: 337-344, 1997-   Niemz, M. H.: Laser-tissue interactions: Fundamentals and    applications. Springer-Verlag, Berlin, 1996-   Oleinick, N. L. and Evans, H. H.: The photobiology of photodynamic    therapy: Cellular targets and mechanisms. Rad. Res. 150: S146-S156,    1998-   Palumbo, G., Caruso, M., Crescenzi, E., Tecce, M. F., Roberti, G.,    and Colasanti, A.: Targeted gene transfer in eukaryotic cells by    dye-assisted laser optoporation. J. Photochem. Photobiol. 36: 41-46,    1996-   Paulus, U., Dreger, P., Viehmann, K., von Neuhoff, N., and Schmitz,    N.: Purging peripheral blood progenitor cell grafts from lymphoma    cells: Quantitative comparison of immunomagnetic CD34⁺ selection    systems. Stem Cells 15: 297-304, 1997-   Pedersen, R. A.: Embryonic stem cells for medicine. Sci. Amer. 280:    68-73, 1999-   Rill, D. R., Santana, V. M., Roberts, W. M., Nilson, T., Bowman, L.    C., Krance, R. A., Heslop, H. E., Moen, R. C., Ihle, J. N., and    Brenner, M. K.: Direct demonstration that autologous bone marrow    transplantation for solid tumors can return a multiplicity of    tumorigenic cells. Blood 84: 380-383, 1994-   Schulze, R., Schulze, M., Wischnik, A., Ehnle, S., Doukas, K., Behr,    W., Ehret, W., and Schlimok, G.: Tumor cell contamination of    peripheral blood stem cell transplants and bone marrow in high-risk    breast cancer patients. Bone Marrow Transplant. 19: 1223-1228, 1997-   Schutze, K. and Lahr, G.: Identification of expressed genes by    laser-mediated manipulation of single cells. Nature Biotechnol. 16:    737-742, 1998-   Sharp, J. G., Joshi, S. S., Armitage, J. O., Bierman, P., Coccia, P.    F., Harrington, D. S., Kessinger, A., Crouse, D. A., Mann, S. L.,    and Weisenburger, D. D.: Significance of detection of occult    Non-Hodgkin's Lymphoma in histologically uninvolved bone marrow by a    culture technique. Blood 79: 1074-1080, 1992-   Sharp, J. G., Kessinger, A., Mann, S., Crouse, D. A., Armitage, J.    O., Bierman, P., and Weisenburger, D. D.: Outcome of high-dose    therapy and autologous transplantation in non-Hodgkin's lymphoma    based on the presence of tumor in the marrow or infused    hematopoietic harvest. J. Clin. Oncol. 14: 214-219, 1996-   Tricot, G., Gazitt, Y., Jagannath, S., Vesole, D., Reading, C. L.,    Juttner, C. A., Hoffman, R., and Barlogie, B.: CD34⁺Thy⁺lin⁻    peripheral blood stem cells (PBSC) effect timely trilineage    engraftment in multiple myeloma (MM). Blood 86: 293a-0, 1995-   Vannucchi, A. M., Bosi, A., Glinz, S., Pacini, P., Linari, S.,    Saccardi, R., Alterini, R., Rigacci, L., Guidi, S., Lombarkini, L.,    Longo, G., Mariani, M. P., and Rossi-Ferrini, P.: Evaluation of    breast tumour cell contamination in the bone marrow and    leukapheresis collections by RT-PCR for cytokeratin-19 mRNA. Br. J.    Haematol. 103: 610-617, 1998-   Vervoordeldonk, S. F., Merle, P. A., Behrendt, H., Steenbergen, E.    J., van den Berg, H., van Wering, E. R., von dem Borne, A. E. G.,    van der Schoot, C. E., van Leeuwen, E. F., and    Slaper-Cortenbach, I. C. M.: PCR-positivity in harvested bone marrow    predicts relapse after transplantation with autologous purged bone    marrow in children in second remission of precursor B-cell acute    leukemia. Br. J. Haematol. 96: 395-402, 1997-   Vredenburgh, J. J., Silva, O., Broadwater, G., Berry, D., DeSombre,    K., Tyer, C., Petros, W. P., Peters, W. P., and Bast, J., R. C.: The    significance of tumor contamination in the bone marrow from    high-risk primary breast cancer patients treated with high-dose    chemotherapy and hematopoietic support. Biol. Blood Marrow    Transplant. 3: 91-97, 1997

1. An apparatus for imaging cells in a biological specimen held in acontainer, comprising: a scanning lens that defines a field of view,wherein said scanning lens is of F-theta design; a computer system thatdivides said field of view into a plurality of frames of cells within abiological specimen, wherein the number of said plurality of frames ofcells within said field of view is between 4 and 24; an illuminationsource for illuminating a frame of cells within said field of view; animage capture system that captures an image of said frame of cells; andcommands for determining a morphological or physiological characteristicof cells in said biological specimen by reference to said image.